Duocarmycin SA (1) and duocarmycin A (2) constitute the parent members of a class of potent antitumor antibiotics related to CC-1065 (3) that derive their properties through a sequence selective alkylation of duplex DNA (FIG. 1). Since their disclosure, substantial efforts have been devoted to defining the characteristics of their DNA alkylation reactions, to determining the origin of their DNA alkylation selectivity, and to defining fundamental relationships between structure, functional reactivity, and biological properties.
Three models have been advanced to account for the DNA alkylation sequence selectivity. One model proposes a sequence-dependent phosphate protonation of the C4 carbonyl which activates the agent for DNA alkylation. (Warpehoski, M. A.; Hurley, L. H. Chem. Res. Toxicol. 1988, 1, 315; Hurley, L. H.; Reynolds, V. L.; Swenson, D. H.; Petzold, G. L.; Scahill, T. A. Science 1984, 226, 843. Reynolds, V. L.; Molineux, I. J.; Kaplan, D. J.; Swedson, D. H.; Hurley, L. H. Biochemistry 1985, 24, 6228. Hurley, L. H.; Lee, C.-S.; McGovren, J. P.; Warpehoski, M. A.; Mitchell, M. A.; Kelly, R. C.; Aristoff, P. A. Biochemistry 1988, 27, 3886. Scahill, T. A.; Jensen, R. M.; Swenson, D. H.; Hatzenbuhler, N. T.; Petzold, G.; Wierenga, W.; Brahme, N. D. Biochemistry 1990, 29, 2852; Hurley, L. H.; Warpehoski, M. A.; Lee, C.-S.; McGovren, J. P.; Scahill, T. A.; Kelly, R. C.; Mitchell, M. A.; Wicnienski, N. A.; Gebhard, I.; Johnson, P. D.; Bradford, V. S. J. Am. Chem. Soc. 1990, 112, 4633; Lin, C. H.; Beale, J. M.; Hurley, L. H. Biochemistry 1991, 30, 3597.) Another invokes alkylation at junctions of bent DNA without addressing the source of catalysis. (Lin, C. H.; Sun, D.; Hurley, L. H. Chem. Res. Toxicol. 1991, 4, 21. Lee, C.-S.; Sun, D.; Kizu, R.; Hurley, L. H. Chem. Res. Toxicol. 1991, 4, 203. Lin, C. H.; Hill, G. C.; Hurley, L. H. Chem. Res. Toxicol. 1992, 5, 167. Ding, Z.-M.; Harshey, R. M.; Hurley, L. H. Nucl. Acids. Res. 1993, 21, 4281. Sun, D.; Lin, C. H.; Hurley, L. H. Biochemistry 1993, 32, 4487. Thompson, A. S.; Sun, D.; Hurley, L. H. J. Am. Chem. Soc. 1995, 117, 2371.) A third model is based on the premise that distinct alkylation selectivities are controlled by the AT-rich noncovalent binding selectivity of the agents and their steric accessibility to the adenine N3 alkylation site. (Boger, D. L.; Johnson, D. S. Angew Chem., Int. Ed. Engl. 1996, 35, 1439. Boger, D. L.; Johnson, D. S. Proc. Natl. Acad. Sci., U.S.A. 1995, 92, 3642. Boger, D. L. Acc. Chem. Res. 1995, 28, 20. Boger, D. L. In Advances in Heterocyclic Natural Product Synthesis; Pearson, W. H., Ed.; JAI: Greenwich, 1992; Vol. 2, 1. Boger, D. L. Chemtracts: Org. Chem. 1991, 4, 329. Boger, D. L. In Proc. R. A. Welch Found. Conf. Chem. Res., XXXV, Chem. Frontiers Med. 1991, 35, 137. Boger, D. L. In Heterocycles in Bioorganic Chemistry; Bergman, J.; van der Plas, H. C.; Simonyl, M., Eds.; Royal Soc. of Chem.: Cambridge, 1991; 103. Coleman, R. S.; Boger, D. L. In Studies in Natural Product Chemistry; Rahman, A.-u.-, Ed.; Elsevier: Amsterdam, 1989; Vol. 3, 301; Boger, D. L.; Johnson, D. S.; Yun, W.; Tarby, C. M. Bioorg. Med. Chem. 1994, 2, 115. Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.; Haught, J.; Bina, M. Tetrahedron 1991, 47, 2661. Boger, D. L.; Coleman, R. S.; Invergo, B. J .; Sakya, S. M.; Ishizaki, T.; Munk, S. A.; Zarrinmayeh, H.; Kitos, P. A.; Thompson, S. C. J. Am. Chem. Soc. 1990, 112, 4623.) This latter proposal accommodates and explains the reverse and offset 5 or 3.5 base-pair AT-rich adenine N3 alkylation selectivities of natural and unnatural enantiomers of duocarmycin and CC-1065 and offers a beautiful explanation for the identical alkylation selectivities of both enantiomers or simple derivatives thereof. Further support for this model includes the demonstrated AT-rich noncovalent binding of the agents, their preferential noncovalent binding coincidental with DNA alkylation, the demonstration that the characteristic DNA alkylation is also observed with isomeric alkylation subunits (e.g., iso-CI and iso-CBI), and that it does not require the presence of the C4 carbonyl or even the activated cyclopropane.
In previous studies, the issue of catalysis with the noncovalent binding model has not been addressed. The chemical stability of duocarmycin and CC-1065 and the acid-catalysis requirement for addition of typical nucleophiles has led to the assumption that the DNA alkylation must also be an acid-catalyzed reaction. Although efforts have gone into supporting the extent and role of this acid catalysis, it remains largely undocumented for the DNA alkylation reaction. At pH 7.4, the DNA phosphate backbone is fully ionized (0.0001-0.00004% protonated). Consequently, it is unlikely that catalysis is derived from a phosphate backbone delivery of a proton to the C4 carbonyl as advanced in the alkylation site model. Consistent with this, the rate of the DNA alkylation reaction for duocarmycin SA exhibits only a very modest pH dependence below pH 7 and essentially no dependence in the more relevant pH 7-8 range.